PROJECT SUMMARY/ABSTRACT Nitrogenase is a complex metalloenzyme that catalyzes one of the most remarkable chemical transformations in biological systems: the nucleotide-dependent reduction of atmospheric dinitrogen to bioavailable ammonia. There is an outstanding interest in decoding the assembly mechanism of nitrogenase, not only because of its significant relevance to nitrogenase-specific reactions, but also because of its tremendous implication for general metalloprotein biogenesis. Here we propose to greatly expand our understanding of the nitrogenase assembly process by combined genetic, biochemical, spectroscopic and structural approaches. The MoFe protein of the molybdenum nitrogenase of Azotobacter vinelandii will be the focus of the proposed investigation. Through our proposed studies, we expect to further refine the biosynthetic pathways of MoFe protein and its associated clusters, a continuous effort with an ultimate goal to unravel the molecular mechanism of nitrogenase assembly. Furthermore, using nitrogenase as a model system, we also hope to provide general themes of biological metal cluster assembly, which may, in the long run, prove instrumental in developing successful strategies for the chemical synthesis of complex metalloclusters. .